title: Integrated mass spectrometry-based analysis of plasma glycoproteins and their glycan modifications.
abstract: We present a protocol for the identification of glycosylated proteins in plasma followed by elucidation of their individual glycan compositions. The study of glycoproteins by mass spectrometry is usually based on cleavage of glycans followed by separate analysis of glycans and deglycosylated proteins, which limits the ability to derive glycan compositions for individual glycoproteins. The methodology described here consists of 2D HPLC fractionation of intact proteins and liquid chromatography-multistage tandem mass spectrometry (LC-MS/MS(n)) analysis of digested protein fractions. Protein samples are separated by 1D anion-exchange chromatography (AEX) with an eight-step salt elution. Protein fractions from each of the eight AEX elution steps are transferred onto the 2D reversed-phase column to further separate proteins. A digital ion trap mass spectrometer with a wide mass range is then used for LC-MS/MS(n) analysis of intact glycopeptides from the 2D HPLC fractions. Both peptide and oligosaccharide compositions are revealed by analysis of the ion fragmentation patterns of glycopeptides with an intact glycopeptide analysis pipeline.
authors.affiliation: Molecular Diagnostics Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. hong.wang.hw3@ventana.roche.com
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Chee-Hong
authors.initials: CH
authors.lastname: Wong
authors.forename: Alice
authors.initials: A
authors.lastname: Chin
authors.forename: Ayumu
authors.initials: A
authors.lastname: Taguchi
authors.forename: Allen
authors.initials: A
authors.lastname: Taylor
authors.forename: Samir
authors.initials: S
authors.lastname: Hanash
authors.forename: Sadanori
authors.initials: S
authors.lastname: Sekiya
authors.forename: Hidenori
authors.initials: H
authors.lastname: Takahashi
authors.forename: Masaki
authors.initials: M
authors.lastname: Murase
authors.forename: Shigeki
authors.initials: S
authors.lastname: Kajihara
authors.forename: Shinichi
authors.initials: S
authors.lastname: Iwamoto
authors.forename: Koichi
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D000465:Algorithms; D020816:Amino Acid Motifs; D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006020:Glycopeptides / Q000097:blood / Q000737:chemistry; D006023:Glycoproteins / Q000097:blood / Q000737:chemistry; D006801:Humans; D011134:Polysaccharides / Q000097:blood / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001798:Blood Proteins; D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides